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The worldwide emergence and spread of antimicrobial resistance in Gram-negative bacteria are severely limiting therapeutic options and thus constitute a major public health threat. The timely accurate detection of carbapenemase producers and the determination of carbapenemase class according to the Ambler classification can guide antimicrobial therapy and facilitate infection control measures. A modified version of the carbapenemase inactivation method (CIM), mCIM, was described and approved by the CLSI in 2017. We evaluated the performance of a faster new mCIM-based assay, mCIMplus, which can detect carbapenemase activity within 8 h and characterize the carbapenemase according to the Ambler classification in 20 h. A panel of 137 isolates producing carbapenemases (GES, IMP, KPC, NDM, OXA-48, OXA-48-like, and VIM enzymes) and 22 non-carbapenemase-producing isolates was used to evaluate the performance of mCIMplus. We evaluated the detection of carbapenemase activity at 8 and 20 h. Carbapenemase class was determined, with specific inhibitors, at 20 h. The sensitivities of mCIMplus were 99.3% at 8 h and 98.5% at 20 h. Its specificity was 100% regardless of culture time. buy EPZ-6438 Based on a decision algorithm, this test successfully identified the carbapenemase class for 98.4% of the tested isolates (127/129). Characterization was correct for 100, 95, and 100% of Ambler class A, B, and D isolates, respectively. This test can, therefore, be used to detect carbapenemase activity within 8 h and to determine carbapenemase class within 20 h. It constitutes a very affordable ( less then €1 per isolate) and reliable technique requiring only basic laboratory equipment.Identification (ID) and antimicrobial susceptibility testing (AST) of respiratory pathogens are critical to the management of patients with pneumonia to facilitate optimal antibiotic therapy selection. Few studies have examined the time to results (TTR) for this critical specimen, and such data can be valuable for benchmarking the current paradigm of diagnostic approaches. TTR for bronchoalveolar lavage (BAL) and endotracheal aspirate (ETA) specimens from hospitalized patients was evaluated using the Premier Healthcare Database, a comprehensive database of 194 U.S. hospitals. Times from specimen collection to reporting of organism ID/AST were evaluated and compared by specimen types and characteristics. A total of 79,662 (43,129 BAL; 36,533 ETA) specimens were included, of which 19.3% harbored no growth, 47.1% contained normal respiratory flora alone (including yeast), and 0.6% contained mycobacteria/molds. Potential bacterial pathogens (PBP) were recovered from 33.0%. ETA specimens had a higher proportion of specimens with isolation of PBP (39.2% versus 27.7%) and with normal respiratory flora (52.0% versus 43.0%) and were less likely to be negative (8.2% versus 28.6%) than BAL specimens (all P less then 0.0001). Staphylococcus aureus and Pseudomonas aeruginosa were isolated in 10.5 and 6.4% of the specimens, respectively, and were the most common organisms identified. Median (interquartile range) TTR were 37.0 h (21.8 to 51.7 h) and 60.5 h (46.6 to 72.4 h) for ID and AST, respectively. Median TTR for major respiratory pathogens by organism ranged from 29.2 to 43.9 h for ID and from 47.9 to 73.9 h for AST. Organism type, specimen collection time, and hospital teaching status influenced TTR. Mechanically vented patients and ETA specimens were more likely to recover PBP.Ancestral genetic exchange between members of many important bacterial pathogen groups has resulted in phylogenetic relationships better described as networks than as bifurcating trees. In certain cases, these reticulated phylogenies have resulted in phenotypic and molecular overlap that challenges the construction of practical approaches for species identification in the clinical microbiology laboratory. Burkholderia cepacia complex (Bcc), a betaproteobacteria species group responsible for significant morbidity in persons with cystic fibrosis and chronic granulomatous disease, represents one such group where network-structured phylogeny has hampered the development of diagnostic methods for species-level discrimination. Here, we present a phylogeny-informed proteomics approach to facilitate diagnostic classification of pathogen groups with reticulated phylogenies, using Bcc as an example. Starting with a set of more than 800 Bcc and Burkholderia gladioli whole-genome assemblies, we constructed phylogenies with explicit representation of inferred interspecies recombination. Sixteen highly discriminatory peptides were chosen to distinguish B. cepacia, Burkholderia cenocepacia, Burkholderia multivorans, and B. gladioli and multiplexed into a single, rapid liquid chromatography-tandem mass spectrometry multiple reaction monitoring (LC-MS/MS MRM) assay. Testing of a blinded set of isolates containing these four Burkholderia species demonstrated 50/50 correct automatic negative calls (100% accuracy with a 95% confidence interval [CI] of 92.9 to 100%), and 70/70 correct automatic species-level positive identifications (100% accuracy with 95% CI 94.9 to 100%) after accounting for a single initial incorrect identification due to a preanalytic error, correctly identified on retesting. The approach to analysis described here is applicable to other pathogen groups for which development of diagnostic classification methods is complicated by interspecies recombination.The objective of this study was to evaluate the Micronaut-S carbapenemase detection microtiter plate assay for the detection of carbapenemases and Ambler class determination. The Micronaut-S carbapenemase detection microtiter plate was tested using a challenging collection of 154 carbapenemase-producing and 150 carbapenemase-negative clinical strains of Enterobacterales and Pseudomonas aeruginosa The Micronaut-S carbapenemase detection assay was able to detect 148/154 carbapenemase producers correctly, whereas 5/150 non-carbapenemase-producing isolates tested as false positive. This resulted in an overall sensitivity of 96% and a specificity of 97%. Regarding the detection of the carbapenemase class, the sensitivities and specificities were 93%/100%, 96%/100%, and 97%/99% for class A (n = 27), class B (n = 54), and class D (n = 73) carbapenemases, respectively. The Micronaut-S carbapenemase detection microtiter plate represents an easy-to-use and valuable tool for accurate and reliable detection of carbapenemases.